Transplantation of cells exhibiting glucose-responsive insulin secretion has the potential to cure diabetes. However, this approach is limited by an inadequate supply of cells with that property, with is exhibited only by pancreatic β-cells. The development of expanded populations of human β-cells that can be used for cell transplantation is therefore a major goal of diabetes research (D. R. W. Group, “Conquering diabetes: a strategic plan for the 21st century” NIH Publication No. 99-4398 (National Institutes of Health, 1999)). A number of alternative approaches are being pursued to achieve that goal, including using porcine tissue as a xenograft (Groth et al., J Mol Med 77:153-4 (1999)), expansion of primary human β-cells with growth factors and extracellular matrix (Beattie et al., Diabetes 48:1013-9 (1999)), and generation of immortalized cell lines that exhibit glucose-responsive insulin secretion (Levine, Diabetes/Metabolism Reviews 1: 209-46 (1997)).
Although there has been great interest in using porcine islets, they are difficult to manipulate in vitro and concerns have been raised about endogenous and exogenous xenobiotic viruses being transmitted to graft recipients (Weiss, Nature 391:327-8 (1998)). With primary human β-cells, entry into the cell cycle can be achieved using hepatocyte growth factor/scatter factor (“HGF/SF”) plus extracellular matrix (“ECM”) (Beattie et al., Diabetes 48:1013-9 (1999), Hayek et al., Diabetes 44:1458-1460 (1995)). However, this combination, while resulting in a 2-3×104-fold expansion in the number of cells, is limited by cellular senescence and loss of differentiated function, particularly pancreatic hormone expression (Beattie et al., Diabetes 48:1013-9 (1999)).
Immortalized cell lines from the human endocrine pancreas have been created to develop β-cell lines that exhibit glucose responsive insulin secretion (Wang et al., Cell Transplantation 6:59-67 (1997), Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)). The cell lines are made by infecting primary cultures of cells from various sources including adult islets, fetal islets, and purified β-cells, with viral vectors expressing the potent dominant oncogenes such as SV40 T antigen and H-rasval12 (Wang et al., Cell Transplantation 6:59-67 (1997), Wang et al., Transplantation Proceedings 29:2219 (1997), Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999); see also U.S. Pat. No. 5,723,333). The combined effect of those oncogenes is to trigger growth factor-independent and extracellular matrix (ECM)-independent entry into the cell cycle, as well as to prolong the lifespan of the cells from 10-15 population doublings or primary cells to approximately 150 doubling for the oncogene-expressing cells (Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)). Further introduction of the gene encoding the hTRT component of telomerase results in immortalization, allowing the cells to be grown indefinitely (Halvorsen et al., Molecular and Cellular Biology 19:1864-1870 (1999)).
Although the cell lines grow indefinitely, they lose differentiated function, similar to growth-stimulated primary β-cells. Methods of stimulating differentiation of the cell lines into insulin-secreting β-cells are therefore desired. Such cells could then be transplanted in vivo as a treatment for diabetes.